Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo.
Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo.
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DOI：
10.1038/nprot.2014.017
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发表时间：
2014-02
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
翻译后修饰改变蛋白质结构，影响活性、稳定性、定位和/或结合伴侣。特异性识别后修饰蛋白质的抗体具有许多用途，包括免疫细胞化学和修饰蛋白质的免疫沉淀以纯化蛋白质-蛋白质和蛋白质-核酸复合物。然而，针对单个蛋白质上的修饰位点的抗体通常是非特异性的。在这里，我们描述了一个协议，以纯化多克隆抗体，特异性地检测修饰的蛋白质的兴趣。该方法使用迭代轮次的减法和亲和纯化，使用严格洗涤来去除识别未修饰的蛋白质和含有修饰的氨基酸的低序列复杂性表位的抗体。采用斑点和蛋白质印迹分析来评估抗体制备特异性。设计该方法以克服单轮消减和亲和纯化不足以获得修饰的蛋白质特异性抗体制剂的常见情况。一轮完整的抗体纯化和特异性检测需要6天的不连续时间。
Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time.
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影响因子：
11.8
作者：
Arur, Swathi;Ohmachi, Mitsue;Berkseth, Matt;Nayak, Sudhir;Hansen, David;Zarkower, David;Schedl, Tim
通讯作者：
Schedl, Tim
影响因子：
2.9
作者：
Ejima, D;Yumioka, R;Arakawa, T
通讯作者：
Arakawa, T
DOI：
10.1073/pnas.94.2.412
发表时间：
1997-01-21
影响因子：
11.1
作者：
Burks, EA;Chen, G;Iverson, BL
通讯作者：
Iverson, BL
DOI：
10.1016/s0165-022x(00)00145-7
发表时间：
2001-03-28
影响因子：
--
作者：
Dansithong, W;Paul, S;Shinozawa, T
通讯作者：
Shinozawa, T
DOI：
10.1016/s1380-2933(96)00045-0
发表时间：
1996-09-01
期刊：
IMMUNOTECHNOLOGY
影响因子：
--
作者：
Davies, J;Riechmann, L
通讯作者：
Riechmann, L